PROJECT SUMMARY The discovery of antibiotics and their introduction into clinical practice has reduced the threat posed by infectious agents and has led to a marked decrease in deaths from diseases that were previously widespread, untreatable and frequently fatal. However, the selective pressure imposed by the widespread use of antimicrobials in human and veterinary medicine has led to the emergence of bacterial strains resistant to first- line therapy. Recognizing the possibility of returning to a clinical equivalent of the pre-antibiotic era, the Federal Government has recently issued an Executive Order (09/18/2014) outlining the immediate need for new antibiotics or alternative therapies to treat infections caused by multi-drug resistant (MDR) pathogens. Lytic bacteriophage are attractive antimicrobials because they are part of the normal human flora, amplify at the site of infection, and are innocuous to human cells. Despite the therapeutic potential of lytic phage, translational and clinical development of natural phage has been hindered mainly by their narrow host specificity compared to antibiotics, poor penetrability into surface attached bacterial communities (biofilm) and susceptibility to both human and bacterial defenses. Because simple methods for manipulation of large virulent genomes are still lacking, most recent efforts to develop highly effective therapeutic phage have largely centered on development of large natural phage cocktails able to eliminate a wide range of bacteria rather than construction of unique phage with desired therapeutic properties. These hurdles can be overcome by genetic engineering of lytic phage. Based on our expertise in assembling and editing large genomes in vitro, our proposal aims to examine the feasibility of a synthetic biology platform for the construction of phage with superior therapeutic efficacy against MDR pathogens. This platform will not only allow rapid expansion of phage host range, but could also be exploited to genetically impart other important characteristics such as biofilm disruption or immunity to host resistance systems. Finally, selected engineered phage strains with desired characteristics will be included in cocktails optimized to kill more than 90% of MDR clinical isolates within a broad library and will be the basis of preclinical product development. Although our platform will have broad applicability for the development of phage as antimicrobials, proof-of-concept studies will focus on MDR P. aeruginosa and its well characterized lytic phage. If successful, this proposal will: i) provide state-of-the-art tools to simplify and accelerate the manipulation and analysis of lytic P. aeruginosa phage, ii) expand the antibacterial spectrum of engineered phage by broadening their host range, iii) provide valuable information on phage-host interactions in an important human pathogen, and iv) create a well-defined and fully characterized bacteriophage cocktail for preclinical development to treat MDR P. aeruginosa.